This invention relates to a method for screening anti-microbial agents, to anti-microbial agents identified by the method, and to a method for combatting microbial growth.
More particularly, the method of the invention relates to the use of a putative ribonucleic acid (RNA) helicase to screen for anti-microbial agents.
Ribonucleic acid (RNA) is an essential molecule for cellular viability in both eukaryotic and prokaryotic organisms. The best known form of RNA is messenger RNA (mRNA) which is essential for protein production as it is the means by which genetic information encoded by the genome in the nucleus of a cell is transported into the cytoplasm for translation into protein. However, other types of RNA are also essential for protein production, namely transfer RNA (tRNA) and ribosomal RNA (rRNA). Ribosomes are the site where protein production actually occurs, by the alignment of a specific tRNA molecule (carrying a specific amino acid) with the corresponding codon (base triplet) on the MRNA. Ribosomes are complex molecules and are known to include several polypeptides as well as rRNA. The rRNA is essential for ribosomal function and is highly conserved between different organisms, although there is a distinct difference between the rRNAs of prokaryotic and eukaryotic ribosomes.
In many RNA molecules function depends on not only the actual sequence of nucleic acid bases (the primary structure), but also upon the manner in which the molecule is folded to form a precise three-dimensional shape (the secondary structure).
Manipulation of RNA secondary structure is an essential step in many key processes in the cell, including RNA splicing, ribosome assembly and translation of mRNA into protein. Thus the RNA helicase enzymes which affect RNA secondary structure are essential for the viability of the cell.
Considerable interest in the phenomenon of RNA secondary structure has been generated by the discovery of a large family of proteins with known, or putative, ATP-dependent RNA helicase activity (Linder et al, 1989; Schmid and Linder, 1992; Fuller-Pace & Lane, 1992). Members of this family form part of a larger superfamily of proteins which interact with polynucleotides and nucleotide triphosphates and share seven highly conserved motifs. In several cases these include a characteristic Asp-Glu-Ala-Asp (DEAD) motif which led to their designation as "DEAD box" proteins (Linder, 1989). However as more proteins were assigned to this family both by sequence homology and in some cases biochemical function, it has become clear that there are at least two subgroups: the DEAD and DExH subgroups (Fuller-Pace, 1992).
Individual members of the DEAD box family have been highly conserved throughout evolution (Linder et al, 1989; Iggo, et al., 1991). The dbpA gene was isolated by hybridisation using a probe from the Saccharomyces pombe homologue of p68, one of the prototypic DEAD box proteins Iggo, et al, 1990). However analysis of the predicted amino acid sequence showed that, although DbpA contains the conserved motifs characteristic of the DEAD box family, it is in fact no more related to p68 than to any other members of the family. Five such genes have been identified in E.Coli (Kalman, et al., 1991). Two of these, srmB (Nishi, et al., 1988) and deaD (Toone, et al., 1991) are thought to be involved in ribosome biogenesis. A third, rhlB, appears to be an essential gene only in some genetic backgrounds (Kalman, et al., 1991) but complementation tests showed no functional complementation between rhlB and srmB, suggesting that these genes have different functions. However little is known about the biological role of rhlB, or rhlE (Kalman, et al., 1991) and dbpA (Iggo, et al., 1990) the other two genes so far identified in E. coli.
The only biochemical study reported to date on any of these E. coli DEAD box proteins has been for the protein SrmB. This protein was shown to hydrolyse ATP in the presence of nucleic acid but it did not show any specificity requirements. The ATPase activity was induced by Poly(U), Poly(A), tRNA, rRNA and to a lesser extent by single stranded and double stranded DNA (Nishi, et al., 1988). Other DEAD box proteins so far examined biochemically, including the prototypic member of the family, the eukaryotic translation initiation factor eIF-4A (Grifo, et al., 1984) also show similar requirements for RNA which are not specific for any particular RNA species in in vitro ATPase assays.
The highly conserved nature of RNA helicases confirms the importance of their function(s), and therefore they offer an attractive target for the action of anti-microbial therapeutic agents.
However, the identification of such agents has been difficult; although therapeutic agents which inactivate a particular RNA helicase of a microbe infecting a patient are theoretically possible, there is always a risk that the RNA helicase of the patient would also be deactivated by the therapeutic agent, resulting in the disabling of vital processes dependent upon the RNA helicase.